The present invention relates to mutants of colony stimulating factors, obtained by recombinant DNA techniques. More specifically, the invention relates to mutants of interleukin-3, containing one or more deletions and/or one or more substitutions, with interesting pharmacological properties.
Historically, factors affecting hematopoietic cells have been detected in an assay measuring the proliferation and/or differentiation of bone marrow cells in soft agar cultures. The factors showing this activity have been collectively called colony-stimulating factors (CSFs). More recently, it has been found that a variety of CSFs exist which, in part, can be classified by the hematopoietic lineages that are stimulated.
In human and murine systems, these proteins include G-CSF and M-CSF. These proteins stimulate the in vitro formation of predominantly neutrophilic granulocyte and macrophage colonies, respectively. Interleukin-2 (xe2x80x9cIL-2xe2x80x9d) stimulates the proliferation of both activated T-cells and activated B-cells, but is not considered a colony stimulating factor.
GM-CSF and interleukin-3 (xe2x80x9cIL-3xe2x80x9d, also known as xe2x80x9cMulti-CSFxe2x80x9d) stimulate the formation of macrophage and both neutrophilic and eosinophilic granulocyte colonies. In addition, IL-3 stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies (D. Metcalf, xe2x80x9cThe hematopoietic colony-stimulating factorsxe2x80x9d, 1984, Elsevier, Amsterdam, and D. Metcalf, Science 299 (1985) 16-22).
Growth factor-induced cell proliferation is a complicated process. Following highly specific binding of the growth factor to its receptor at the cell surface, the complex is internalized by endocytosis and induces an intracellular response often preceded by phosphorylation of the receptor (Sibley e al., Cell 48 (1987) 913-922). These intracellular signals result in specific gene transcription and finally in DNA synthesis and cell replication.
There is considerable interest in the CSFs, since they may be therapeutically useful for restoring depressed levels of hematopoietic and lymphoid stem cell-derived cells.
Human IL-3 (xe2x80x9chIL-3xe2x80x9d) is such a CSF. Mature hIL-3 consists of 133 amino acids; the protein contains one disulfide bridge and has two potential glycosylation sites (Yang et al., Cell 47 (1986) 3-10). It has inter alia the following activities:
1) stimulation of colony formation by human hematopoietic progenitor cells wherein the colonies formed include erythroids, granulocytes, megakaryocytes, granulocyte macrophages, and mixtures thereof; and
2) stimulation of DNA synthesis by human acute myelogenous leukemia (AML) blasts.
Useful agonists and antagonists of a proteir can be created once the structure-function relationship of the molecule is understood. Generally, this relationship is studied by modifying, replacing or deleting amino acids. In this way, information can be obtained about the importance of each of the amino acids for the activity of the protein. Important domains of proteins may be the active site, metal and cofactor binding sites, receptor binding sites, the amino acids involved in subunit interactions, and the antigenic determinants.
Once the primary sequence of a protein has been determined, various procedures can be employed to study the above-mentioned characteristics. For example, if primary structures of homologous proteins from other species are available, the sequences can be compared. Conserved sequences are often indicative of the importance of certain amino acids.
Secondary structures can be predicted with the use of known algorithms. See, e.g., Hopp and Woods, Proc. Natl. Acad. Sci. USA 78 (1981) 3824-3828, Garnier et al., J. Mol. Biol. 120 (1978) 97-120, Biou et al., Prot. Eng. 2 (1988) 185-191, Carmenes et al., Biochem. Biophys. Res. Commun. 159 (1989) 687-693.
If interspecies homology between homologous proteins is high and the 3-D structure of one of them is known, important amino acids can also be deduced from this structure.
Primary and/or spatial-structure data can be used to make an educated guess for mutagenesis experiments. Expression of mutagenized proteins and the testing of these muteins in biological assays provides information about the relative importance of certain amino acids.
The aim of the present invention is to provide IL-3 mutants with similar or improved pharmaceutical properties with respect to the native IL-3, preferably using the procedures mentioned above.
In 1984, cDNA clones coding for murine IL-3 were isolated (Fung et al., Nature 307 (1984) 233-237 and Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074). This CDNA would not hybridize with human DNA or cDNA clones. Thus, it was speculated that a human counterpart for murine IL-3 (mIL-3) did not exist. This belief was reinforced by the wide spectrum of activities of the human GM-CSF. Finally, in 1986, a gibbon cDNA expression library provided the gibbon IL-3 sequence. This sequence was subsequently used as a probe against a human genomic library. This provided evidence for the presence of IL-3 in human beings (Yang et al., Cell 47 (1986) 3-10).
Meanwhile, Dorssers et al., Gene 55 (1987) 115-124, found a clone from a human cDNA library that surprisingly hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3xe2x80x2 noncoding regions of mIL-3 and hIL-3.
Moonen e al., Proc. Natl. Acad. Sci. USA 84 (1987) 4428-4431 describe the production of human GM-CSF by several recombinant sources including E. coli, yeast and animal cells. Partially purified expression products from yeast and animal cells were assayed for the effect of deglycosylation. The immunoreactivity was increased 4- to 8-fold upon removal of the N-linked oligosaccharides. The specific biological activity was increased by a factor of 20, both in the chronic myelogenous leukemia (CML) and in the human bone marrow assay.
Kaushansky e a., Proc. Natl. Acad. Sci. USA 86 (1989) 1213-1217, tried to define the region(s) of the GM-CSF polypeptide required for biological activity. Since human and murine GM-CSF do not cross-react in their respective colony-forming assays, the approach was based on the use of hybrid DNA molecules containing various lengths of the coding regions for h- and mGM-CSF. After expression in COS cells, the hybrid proteins were tested in both human and murine colony-forming assays. Two regions of GM-CSF were found to be critical for hematopoietic function. These regions are structurally characterized by an amphiphilic helix and by a disulfide-bonded loop.
Gough et al., Eur. J. Biochem. 169 (1987) 353-358, describe internal deletion mutants of murine GM-CSF. None of the mutants is reported to show biological activity.
Kuga et al., Biochem. Biophys. Res. Com. 159 (1989) 103-111, describe mutagenesis of human G-CSF. The results indicate that most of the expression products with mutations localized in the internal or C-terminal regions abolish hG-CSF activity. On the other hand, N-terminal deletion mutants missing 4, 5, 7 or 11 amino acids, out of a total of 174 amino acids, retained activity. Some of the N-terminal amino acid mutants showed increased activity.
Deletion mutants of human interleukin-1 (IL-1) have been created using available endonuclease restriction sites and expression in eukaryotic cells. The carboxyl terminal third (63 amino acids) of the polypeptide contains the active site (Makino et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7841-7845). A recent study on IL-1alpha and IL-1beta shows that 140 and 147 amino acids, respectively (out of a total of 153 amino acids), are required for full biological activity (Mosley et al., Proc. Natl. Acad. Sci. USA 84 (1987) 4572-4576). Single amino acid changes at both termini result in significant decrease of biological activity. However, no detailed information with respect to the receptor-binding domain of IL-1 has been obtained from these studies.
Activity of human interleukin-2 was shown to be severely inhibited by removal of both Cys58 and Cys105 whereas deletion of the third Cys residue (125) had no effect (Wang et al., Science 224 (1984) 1431-1433; Cohen et al., Science 234 (1986) 349-352). All substitutions resulting in a disturbance of helical folding of this protein were found to give significant reductions of biological activity. The potential receptor binding site of IL-2 has been mapped on an eleven amino acid long peptide. Individual amino acid substitutions in this region had dramatic effects (Cohen et Al., (supra); Zurawski and Zurawski, EMBO J. 7 (1988) 1061-1069). Mutational analysis further revealed that different domains of IL-2 are involved in high and low affinity binding of the IL-2 receptor complex (Robb et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5654-5658; Collins et al., Proc. Natl. Acad. Sci. USA 85 (1988) 7709-7713).
Clark-Lewis et Al., Science et al., (1986) 134-139, performed a functional analysis of synthetic murine IL-3 analogs. They concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of two of the four Cys residues by Ala (Cys79, Cys140xe2x86x92Ala79, Ala140) resulted in increased activity (Clark-Lewis et al., Proc. Natl. Acad. Sci. USA 85 (1988) 7897-7901).
Literature on proposed and actually performed modifications of hIL-3 is scarce. International Patent Application (PCT) WO 88/00598 discloses a Ser27xe2x86x92Pro27 replacement. (It should be noted that the numbering of amino acids in WO 88/00598 includes the signal peptide of 19 amino acids.) Furthermore, suggestions are made to replace Cys with Ser, breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites (Asn34cys35Ser36 and Asn89Ala90Ser91).
EP-A-0275598 (WO 88/04691) illustrates that Ala1 can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, viz. two double mutants, Ala1xe2x86x92Asp1, Trp13xe2x86x92Arg13 (pGB/IL-302) and Ala1xe2x86x92Asp1, Met3xe2x86x92Thr3 ((pGB/IL-304) and one triple mutant Ala1xe2x86x92Asp1, Leu9xe2x86x92Pro9, Trp13xe2x86x92Arg13 (pGB/IL-303).
WO88/05469 describes deglycosylation mutants and mutants of Arg54Arg55 and Arg108Arg109Lys110 (converted to the same numbering as in EP-A-0275598). The latter are suggested in order to avoid proteolysis upon expression in Saccharomyces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast.
Finally, EP-A-0282185 describes various mutants that may be conformationally and antigenically neutral. To achieve this, a series of synonymous amino acid substitutions are suggested. The proposed changes are aimed at keeping the structure and charge distribution of the IL-3 molecule unaltered. However, the only actually performed mutations are Met2xe2x86x92Ile2 and Ile131xe2x86x92Leu131. It is not disclosed whether the contemplated neutralities are obtained.
No known extensive mutagenesis experiments on hIL-3 have been disclosed to date.
The present invention provides new classes of pharmacologically interesting compounds, viz. deletion and substitution mutants of hIL-3, showing biological activities similar and in some cases possibly antagonistic to those of hIL-3.
In one aspect of the invention, biologically active polypeptide analogs of human interleukin-3 (also referred to hereinafter as xe2x80x9chIL-3 mutantsxe2x80x9d or xe2x80x9cmuteinsxe2x80x9d) are provided having a deletion of at least two amino acids.
Preferred mutants are those having one or more deletions at the N-terminus (amino acids 1-14) and/or the C-terminus (amino acids 120-130 and/or 130-133).
In another aspect of this invention, substitution mutants of hIL-3 are disclosed having at least one of the following substitutions:
Asp21Glu22xe2x86x92Lys21Arg22 
Asp36xe2x86x92Arg36 
Glu43Asp44xe2x86x92Lys43Arg44 
Arg54Arg55xe2x86x92Glu54Asp55 
Asp46xe2x86x92Lys46 or Arg46 
Glu50xe2x86x92Lys50 or Arg50,
Glu59xe2x86x92Lys59 or Arg59 
Glu59xe2x86x92Gly59 or Pro59,
Arg63Ala64xe2x86x92Pro63Gly64 
Glu75xe2x86x92Arg75 or Gly75 
Lys79xe2x86x92Glu79 
Arg94xe2x86x92Pro94 
His98Lys100Asp101xe2x86x92Glu98Asp100Gln101 
Glu106xe2x86x92Lys106 
Arg108Arg109Lys110xe2x86x92Glu108Asp109Glu110,
Phe113Tyr114xe2x86x92Ala113Thr114 
Cys16xe2x86x92Ala16 
Cys84xe2x86x92Ala84 
Cys16 Cys84xe2x86x92Ala16 Ala84 
In yet another aspect of this invention, antagonists of hIL-3 are disclosed. These antagonists are substitution mutants of hIL-3 that are more potent in receptor binding than in stimulation of DNA synthesis. More specifically, the antagonists are single or double Cys mutants. Preferably Cys is replaced by Ala (Cys16xe2x86x92Ala16, Cys16Cys84xe2x86x92Ala16Ala84). Other mutants having an antagonistic effect are Glu50xe2x86x92Lys50 and Lys79xe2x86x92Glu79.
The polypeptides are obtained through expression of suitably modified DNA sequences. Thus, the present invention also provides suitable expression vectors and host cells compatible therewith.
In yet other aspects, the invention comprises pharmaceutical compositions that include biologically active peptide analogs of hIL-3 as described above, in combination with a pharmaceutically acceptable carrier.
Finally, the present invention discloses monoclonal antibodies aimed at an epitope localized between amino acids 29 and 54.
Other embodiments of the subject invention are readily determined by one skilled in the art.